ISOLATION OF GUS+VE TRANSCONJUGANTS OF RHIZOBIUM

MAY-JUNE 1996

SPIC SCIENCE FOUNDATION

GUIDE: Dr. WAHEEDA HOPPER

SUMMER RESEARCH AS AN UNDERGRADUATE

My work was part of a project which the Microbiology Department was involved with. The aims of the project were:

Ø To introduce Nitrogen fixing genes into non-leguminous corps like rice, maize, wheat.

Ø To produce a biological N2 fertilizer that would be bio-degradable.

My work involved introducing the transposons carrying the gusA marker gene (encoding b-glucuronidase) into a recipient Rhizobium strain. The transposons were maintained on Hfr E.coli plasmid and transfer was achieved through bacterial conjugation. Conjugation was carried out by mixing in non-selective conditions and growth was allowed in selective medium for transconjugants. Selection was based on the b-glucuronidase enzyme system. Blue colonies were selected from IPTG/Xgal plates. The transposon also carried the aadA gene encoding resistance to streptomycin and spectinomycin. 3 transposons namely:

Ø mTn5SSgusA11 & mTn5SsgusA20 with constitutive promoters were used to study rhizobial competition

Ø mTn5SsgusA40 where gusA gene expression is dependent on host genetic control was used to create strains that produce gus only in response to specific signals e.g., root exudate.